Dr. Daggett requests continuation support to perform computer simulations of protein denaturation in solution. Considerable progress with unfolding simulations during the last funding period is described, with respect to establishing the methods, devising ways to identify and test models of transition states, developing analysis tools for the evaluation of residual structure in partially unfolded and denatured proteins, developing approaches for comparison of simulation results with experiment, and predicting experimental outcomes. But, there are still a number of issues that need to be addressed. High temperature molecular dynamics simulations of the unfolding of small globular proteins will continue continue, but huge differences between the experimental time scale of protein unfolding and what is accessible with current computer power. The validity of this approach will be evaluated by performing simulations at a variety of temperatures, using chemical denaturants, and performing temperature-quenched simulations. Also, while it is desirable to have the ability to follow a single protein over time, now multiple simulations will be performed to ensure that the effects observed are representative of the much larger ensemble probed experimentally, and the transition state ensembles identified from the simulations tested rigorously, to ensure that they are reasonable. The project will be extended to free energy perturbation calculations to provide more direct comparisons with experimental results. Finally, mutant proteins with altered folding behavior are being designed, based solely on the molecular dynamics-generated models. The mutants are being produced and experimentally evaluated in the lab of a collaborator, Prof Alan Fersht, an eminent Cambridge UK enzymologist.